Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues

  • Mireille Faurobert
  • Esther Pelpoir
  • Jamila Chaïb
Part of the Methods in Molecular Biology book series (MIMB, volume 355)


Phenol extraction of proteins is an alternative method to classical TCA-acetone extraction. It allows efficient protein recovery and removes nonprotein components in the case of plant tissues rich in polysaccharides, lipids, and phenolic compounds. We present here a tried and tested protocol adapted for two dimensional electrophoresis (2-DE) and further proteomic studies. After phenol extraction, proteins are precipitated with ammonium acetate in methanol. The pelleted proteins are then resuspended in isoelectric focusing buffer, and the protein concentration is measured with a modified Bradford assay prior to electrophoresis.

The important points for successful use of this protocol are (1) keeping samples at very low temperature during the first step and (2) careful recovery of the phenolic phase after the centrifugations, which are major features of this protocol.

Key Words

extraction method proteins phenol plant proteomic membrane proteins two-dimensional gel electrophoresis glycoproteins 


  1. 1.
    Michaud, D. and Asselin, A. (1995) Application to plant proteins of gel electrophoretic methods. J. Chromatogr. A 698, 263–279.CrossRefGoogle Scholar
  2. 2.
    Damerval, C., Zivy, M., Granier, F., and de Vienne, D. (1988) Two-dimensional electrophoresis in plant biology. Adv. Electrophoresis 2, 263–340.Google Scholar
  3. 3.
    Hurkman, W. J. and Tanaka, C. K. (1986) Solubilisation of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806.CrossRefPubMedGoogle Scholar
  4. 4.
    Mijnsbrugge, K. V., Meyermans, H., Van Montagu, M., Bauw, G., and Boerjan, W. (2000) Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins. Planta 210, 589–598.CrossRefGoogle Scholar
  5. 5.
    Mihr, C. and Braun, H. P. (2003) Proteomics in plant biology, in Handbook of Proteomics (Conn, P., ed.), Humana Press, Totowa, NJ, pp. 409–416.CrossRefGoogle Scholar
  6. 6.
    Carpentier, S. B., Witters, E., Laukens, K., Deckers, P., Swennen, R., and Panis, B. (2005) Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. Proteomics 5, 2497–2507.CrossRefPubMedGoogle Scholar
  7. 7.
    Wang, W., Scali, M., Vignani, R., et al. (2003) Protein extraction for two-dimensional electrophoresis from olive leaf, a plant tissue containing high levels of interfering compounds. Electrophoresis 24, 2369–2375.CrossRefPubMedGoogle Scholar
  8. 8.
    Saravanan, R. S. and Rose, J. K. C. (2004) A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics 4, 2522–2532.CrossRefPubMedGoogle Scholar
  9. 9.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.CrossRefPubMedGoogle Scholar
  10. 10.
    Lowry, H., Rosebrough, J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.PubMedGoogle Scholar
  11. 11.
    Ramagli, L. S. and Rodriguez, L. V. (1985) Quantification of microgram amounts of protein in two dimensional polyacrylamide gel electrophoresis sample buffer. Electrophoresis 6, 559–563.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Mireille Faurobert
    • 1
  • Esther Pelpoir
    • 1
  • Jamila Chaïb
    • 1
  1. 1.UR de Génétique et Amélioration des Fruits et LégumesINRA AvignonMontfavetFrance

Personalised recommendations